Co-Expression of JcNAC1- and JcZFP8-Improved Agronomic Traits of Tobacco and Enhanced Drought Resistance through NbbHLH1 and NbbHLH2

Previous studies have identified numerous transcription factors involved in drought response, each of which play different roles in plants. The objective of the present study was to evaluate the effectiveness of two transcription factors on drought response in Jatropha curcas L., JcNAC1 and JcZFP8. The overexpression of these transcription factors in tobacco (Nicotiana benthamiana L.) improved drought resistance, but JcZFP8 delayed germination and JcNAC1 reduced biomass and yield. By constitutively co-expressing these two genes in tobacco, drought resistance was improved, and the negative effects of each of them were overcome. The transgenic plants with double-gene co-expression showed stronger drought tolerance with 1.76-fold greater accumulation of proline and lower H2O2 and malondialdehyde (MDA) content to 43 and 65% of wildtype (WT) levels, respectively. The expression levels of NbbHLH1 and NbbHLH2 genes upregulated linearly with the increased drought tolerance of double genes co-expression plants. In drought conditions, the leaf water contents of bhlh1, bhlh2, and bhlh1bhlh2 deletion mutants obtained by CRISPR-CAS9 knockout technique were maintained at 99%, 97%, and 97% of WT. The bhlh1bhlh2 was found with lower germination rate but with higher reactive oxygen levels (1.64-fold H2O2 and 1.41-fold MDA levels). Thus, the co-expression of two transcription factors with different functions overcame the adverse traits brought by a single gene and enhanced the shared drought-tolerant traits, which can provide guidance on theory and selection of gene combinations for the application of multi-gene co-expression in agriculture in the future.


Introduction
Drought stress affects plant survival and growth.To explore this adverse effect, scientists seek to breed highly drought-tolerant crops by transgenic technology or genetic modification methods [1,2].However, different drought tolerance genes have different effects on plant drought resistance, and the strategy for using them in response to drought stress must take these differences into account.Drought tolerance genes are often associated with unfavorable plant growth traits, such as growth inhibition [3][4][5].
NAM/ATAF/CUC protein (NAC) and C2H2 zinc finger protein (ZFP) are involved in plant growth and stress responses [6,7].VaNAC17 improves drought tolerance in Arabidopsis by upregulating genes involved in JA signaling pathways and enhancing ROS scavenging [8].OsZFP15 reduces sensitivity to ABA and improves drought tolerance in rice [9].OsDRZ1 enhances the drought resistance of rice by enhancing antioxidant capacity [10].The basic helix-loop-helix (bHLH) is involved in plant drought responses [11].NbbHLH1 and NbbHLH2 function as positive regulators in the jasmonate signal transduction pathway [12].AhbHLH112 enhances ROS-scavenging ability by regulating Peroxidase (POD)-mediated H 2 O 2 homeostasis [13].PtrbHLH66 improves the drought tolerance in Arabidopsis by increasing the proline contents and antioxidant enzyme activities, and reducing reactive oxygen species (ROS) and malondialdehyde (MDA) under drought stress [14].
Drought is a polygenic trait, and potential candidate genes contribute to cell detoxification, osmotic accumulation, antioxidant mechanisms, and signaling pathways [15].The overexpression of both OsPIL1 and AtDREB1A in Arabidopsis improves drought tolerance and plant growth [16].The co-expression of AtGA5 and AtDREB1A increases Arabidopsis biomass and flower induction, and leads to high levels of drought stress tolerance [17].The co-expression of NHX1 and eIF4A1 from Arabidopsis positively regulates drought stress tolerance in sweet potato [18].However, single gene transformation cannot meet our needs for drought tolerance and agronomic traits, and the co-expression of multiple genes is currently a major challenge in biogenetic engineering.Co-expressing stress response genes and growth regulatory genes can compensate for the accumulation of trade-off genes.This strategy can be effective in reducing adverse growth traits by the overexpression of stress response genes, but the mechanism of this strategy is extremely complex and less research has been conducted in this area [15,19].
Multiple research projects on drought resistance of transcription factors have mainly focused on a single transcription factor, while few studies have thoroughly investigated on the regulation of plant growth traits and drought resistance by co-expression of double transcription factors.JcNAC1 and JcZFP8 are two different transcription factors cloned from Jatropha curcas L. in our previous studies [20,21].Experimental approaches transferred JcNAC1 (NAC) and JcZFP8 (ZFP) into Nicotiana tabacum L., and the results confirmed that plants with double gene co-expression (NZ) had preferable growth traits and drought tolerance.Using qRT-PCR and CRISPR-Cas9, we found that JcNAC1 and JcZFP8 enhanced the expression of NbbHLH1 and NbbHLH2, and the knocking out tobacco of NbbHLH1 and NbbHLH2 reduces tolerance to drought.This combined pattern of transcription factors has potential applications in crop improvement programs.We sought to determine the germination rate under the stress of low water potential (physiological drought), which we imposed using a high concentration of the osmoticum mannitol.When the mannitol concentration was 150 mM and 200 mM, the relative germination rates of NAC, ZFP, and NZ overexpression lines were significantly higher than WT (Figure 1a and Table S1).Increasing the mannitol concentration from 150 to 200 mM decreased the germination rate about 44% in WT, but only decreased it by about 17% in the overexpression lines.This suggests that the tolerance of NAC, ZFP, and NZ to mannitol-drought was higher than that of WT.

Co-Expression of JcNAC1
To understand the seed germination ability of transgenic plants, the number of germinated seeds was counted daily from the beginning of inoculation (Figure 1b).All lines started to germinate on day 3.Among them, the double overexpression line NZ had the fastest germination rate, followed by WT, NAC, and ZFP.According to the calculation of the germination index of the seeds of each strain, NZ had the highest germination index, and the germination index of NAC and ZFP were lower than WT (Figure 1c and Table S2), which indicated that the overexpression of JcNAC1 and JcZFP8 delayed seed germination.The results with the two-gene overexpression line NZ suggested that there was an interaction between two genes that enhanced seed germination rate.
The results with the two-gene overexpression line NZ suggested that there was an interaction between two genes that enhanced seed germination rate.Transgenic lines improved seed germination under osmotic stress.Five-week-old tobacco treated with 10% PEG6000 for 4 days showed significant differences between WT and transgenic tobacco.WT showed obvious wilting, whereas the leaves of the overexpression lines of tobacco showed only slight wilting (Figure 1d).
Stomata are the structures that regulate plant transpiration.Under stress, the stomata of WT closed on day 2, and remained closed through day 8, while the stomata of NAC, ZFP, and NZ plants closed more slowly and had significantly higher stomatal conductance on day 2 than WT (Figure 1e and Table S3).Correspondingly, transpiration rate of WT decreased fastest and transpiration rate of NAC, ZFP, and NZ decreased more slowly (Figure 1f and Table S3).
The photosynthetic rate of WT decreased to near zero at day 2 of PEG osmotic stress whereas the overexpression lines maintained high photosynthetic rates at day 2, and the ZFP and NZ overexpression lines maintained high photosynthetic rates at day 4 (Figure 1g and Table S3).ZFP and NZ had higher photosynthetic rates than WT at day 6 and day 8 of stress as well.

Double Gene Co-Expressing Plants Overcome the Adverse Agronomic Traits Brought by Single Gene Transformation
For the changes in germination of transgenic lines, we studied the growth traits of transgenic lines.At three weeks after planting, ZFP lines had only developed about 3.5 expanded leaves, whereas WT, NAC, and NZ lines had developed 4.5 leaves.Upon observing the leaf size, we found that ZFP had smaller leaves.Fewer and smaller leaves indicated ZFP's early stage growth inhibition (Figures 1h and S1).
Upon observing the flowering time of different plant lines, we found that the average flowering time from early to late was NAC, NZ, WT, and ZFP.The flowering period of NAC was 3 days earlier than WT (Figure 1i and Table S2).The average life cycle of NAC was 4 days shorter than that of WT and ZFP (Figure 1j and Table S2).However, in NZ, while flowering was intermediate between wildtype, ZFP, and NAC, the duration of the life cycle was short, as in NAC.
After harvesting the plants that had completed the life cycle, the biomass and yield of the different plants were statistically analyzed.The results showed that the average biomass of NZ and ZFP was significantly higher than WT and NAC, and the biomass of NZ was 16% higher than WT (Figure 1k and Table S2).The seed yields of NZ and ZFP were also significantly higher than WT, while the seed yield of NAC was lower than WT and the yield of NZ was 14% higher than WT (Figure 1l and Table S2).In conclusion, NZ obtained higher biomass accumulation and seed yield, suggesting that co-over-expression of the two genes in tobacco NZ may have overcome the germination delay and growth inhibition phenotype in early stage caused by the overexpression of JcZFP8, and the reduction in biomass and yield caused by the overexpression of JcNAC1.

More Proline and Less Reactive Oxygen Species (ROS) Level Endows the Double Gene Overexpression Lines with Stronger PEG-Drought Resistance 2.2.1. Under 10%PEG6000 Treatment, the Accumulation of ROS in Transgenic Plants Was Less
In this study, five-week-old plants were treated with PEG6000 at 10% concentration, to compare the ROS levels of WT and transgenic plants after 4 days of stress.NBT and DAB staining were used to provide a qualitative measure of the levels of O 2− and H 2 O 2 (Figure S2a,b).According to the degree of leaf staining, it appeared that ROS accumulation in NAC, ZFP, and NZ was less than in WT.A quantitative assay of O 2− and H 2 O 2 levels indicated that WT accumulated considerably higher concentrations of ROS than the overexpression lines; the accumulated O 2− of ZFP was higher than that of NAC and NZ (Figure 2a and Table S4), and the H 2 O 2 content of NAC was higher than that of ZFP and NZ (Figure 2b and Table S4).The content of O 2-and H 2 O 2 in NZ was 33% and 43% of that in WT.
DAB staining were used to provide a qualitative measure of the levels of O 2− and H2O2 (Figure S2a,b).According to the degree of leaf staining, it appeared that ROS accumulation in NAC, ZFP, and NZ was less than in WT.A quantitative assay of O 2− and H2O2 levels indicated that WT accumulated considerably higher concentrations of ROS than the overexpression lines; the accumulated O 2− of ZFP was higher than that of NAC and NZ (Figure 2a and Table S4), and the H2O2 content of NAC was higher than that of ZFP and NZ (Figure 2b and Table S4).The content of O 2-and H2O2 in NZ was 33% and 43% of that in WT.Proline concentration was lowest in WT, and was considerably higher in the overexpression lines (Figure 2c and Table S4).The accumulation of proline in NZ plants was 1.76 times in WT and higher than that in NAC and ZFP.
At under 10% PEG6000 stress, the MDA content in WT was the highest, and was significantly lower in the other lines (Figure 2d and Table S4).The MDA accumulation of NZ Proline concentration was lowest in WT, and was considerably higher in the overexpression lines (Figure 2c and Table S4).The accumulation of proline in NZ plants was 1.76 times in WT and higher than that in NAC and ZFP.
At under 10% PEG6000 stress, the MDA content in WT was the highest, and was significantly lower in the other lines (Figure 2d and Table S4).The MDA accumulation of NZ plants was 65% of that of WT; the oxidative damage in NZ and ZFP was less than that in WT and NAC.
In conclusion, under drought stress, the co-expressed tobacco NZ had more proline and less ROS, which likely contributed to its tolerance of osmotic stress.

NbbHLH1 and NbbHLH2 Genes Increased Linearly from WT to Single-Overexpression Lines to NZ under PEG-Drought
To further test the relationship between the transcription factors JcNAC1 and JcZFP8 in hormone signaling, we analyzed the expression of several key genes in the ABA, JA, and BR signaling pathways and the regulation of downstream transcription factors by qRT-PCR (Figure 2e).The expression of several of these genes was substantially higher in NZ than in WT and the other lines, while the single-gene overexpressed lines tended to have only slightly increased or the same level of expression as the WT.Those in which NZ was substantially higher than the other lines included: the ABA-related genes NbSNRK2E and NbSNRK2.7, the JA-related genes NbCOI1 and NbJAZ, the BR-related genes NbBRI1 and NbBSK, the NAC gene NbNAC25, the MYB-related genes NbMYB44 and NbMYB86, the ZFP gene NbZFP8, the ERF-related genes NbERF3 and NbDREB2a, the WRKY gene NbWRKY40, and the bHLH genes NbbHLH1 and NbbHLH2 (Figure 2g).Overall, the expression of other transcription factors was changed, but there was no obvious rule.The expression levels of NbbHLH1 and NbbHLH2 genes increased linearly from WT to single-overexpression lines to NZ.To further explore the effect of NbbHLH1 and NbbHLH2 on the drought-resistant characteristics of tobacco, we selected WT for gene knockout verification The CRISPR knockout technique was used to study the role of NbbHLH1, NbbHLH2 in drought tolerance.The vectors PHSE401-bHLH1A1B1, PHSE401-bHLH2A2B2, and PHSE401-bHLH1B2-bHLH2B2 were constructed.The mutants of bhlh1, bhlh2, and bhlh1bhlh2 were obtained (Figures S4 and S5).
The germination rate of the three knockout lines in 150 mM and 200 mM mannitol medium was substantially lower than in 0 mM mannitol medium, but in 150 and 200 mM mannitol, the knockout lines were only slightly lower, though significantly (p ≤ 0.05) than the WT (Figure 3a,b and Table S5).The knockout lines at five weeks of age were stressed with withholding watering for 7 days.The relative water contents of bhlh2 and bhlh1bhlh2 leaves were significantly lower than in the WT, although the magnitude of the effect was relatively small (Figure 3c and Table S6).The relative water content of in bhlh1, bhlh2, and bhlh1bhlh2 was 99%, 97%, and 97% of that in WT, respectively.

H 2 O 2 and MDA Was Increased in bhlh1, bhlh2, and bhlh1bhlh2
The knockout lines at five weeks of age were stressed with withholding watering for 7 days, MDA and H 2 O 2 contents of mutant tobacco were higher than WT, indicating that bhlh1, bhlh2, and bhlh1bhlh2 accumulate more ROS.(Figure 3d,e and Table S6).By comparing single knockout tobacco with double knockout tobacco, it was found that the H 2 O 2 content of bhlh2 was lower than that of bhlh1 and bhlh1bhlh2, while the contents of MDA were higher than WT in all three mutants.The contents of MDA and H 2 O 2 in the leaves of bhlh1bhlh2 were 1.41 and 1.64 times that of WT, indicating that the ROS scavenging ability of bhlh1bhlh2 tobacco was weaker than that of WT.Since NbbHLH1 and NbbHLH2 are important response genes downstream of JA signal transduction, we determined the regulation of the expression of key JA signaling genes in WT and mutant tobacco under drought conditions (Figure 3f).All three knockout lines had a lower expression than WT of NbJAR1, NbCOI1, and NbJAZ, but the expression of NbJA3 was only slightly lower in bhlh1 and bhlh1bhlh2 than in WT.We analyzed the expression of MYB-type transcription factors in the knockout lines and found that two genes, NbMYB21 and NbMYB86, were downregulated only in bhlh1 mutants (Figure 3g).There was no significant difference in expression in the other two knockout lines compared with the WT, suggesting that NbbHLH1 and NbbHLH2 might not regulate the drought response by affecting the expression of NbMYB21 and NbMYB86.
In conclusion, knock-out tobacco bhlh1, bhlh2, and bhlh1bhlh2 had lower germination rate under mannitol, more MDA and H 2 O 2 under drought, which indicates its weakened tolerance to drought stress.

Discussion
Drought stress is the most catastrophic stress affecting crops that has a serious impact on crop yield [22].Most of the research on the application of transcription factors to drought resistance has focused on the effect of individual transcription factors on the resistance of plants [23].The overexpression of a transcription factor alone can enhance drought resistance while often associating adverse growth traits such as retarded growth [5].There are few reports of co-expression of different transcription factors to produce better growth and drought resistance traits in plants.We characterized JcNAC1 and JcZFP8 co-expression and found that it can overcome the unfavorable growth traits of single gene plants and engender stronger drought resistance.JcNAC1 and JcZFP8 synergistically enhanced the expression of NbbHLH1 and NbbHLH2 to improve the drought resistance of plants.This gene combination had a good balance between growth traits and stress resistance, which has great potential in agriculture.
Studies have shown that stress-induced promoters are crucial for achieving ideal expression of transcription factors, which can eliminate the damage of constitutive expression on plant growth and development [24].By using the Oshox24 promoter, AtDREB1A can enhance rice drought resistance through stress-induced expression while relieving the growth inhibition caused by constitutive expression [25].Prd29A:TaDREB2B transgenic sugarcane can enhance drought resistance without affecting growth [26].The inducible composition of pAsr11875:SaADF2 in Arabidopsis enhances drought resistance while relieving the growth inhibition caused by constitutive expression [24].In our study, both JcNAC1 and JcZFP8 were constitutively expressed and not only enhanced tobacco drought-resistance but also affected plant growth.The use of drought-induced promoters to achieve the induced expression of JcNAC1 and JcZFP8, which can improving plant drought resistance without affecting plant growth, was considered.The strategy can be considered but requires further research.
The current studies showed that single-gene overexpression of JcNAC1 or JcZFP8 in tobacco decreased germination index, but the co-expression of these genes in NZ stimulated germination index to levels higher than WT (Figure 1c).In Arabidopsis, gibberellin (GA) can mediate endosperm expansion and regulate seed germination, and RGL2 in the GA pathway repressed activation of the EXPA2 promoter by NAC25/NAC1L, while NAC1L has been identified as an upstream regulator of EXPA2 expression [27].JcZFP8 affects tobacco plant height via GA [21].According to these studies, the co-expression of JcNAC1 and JcZFP8 may play a role in the GA/DELLA-NAC-EXPA2 network, to promote seed germination.However, how these two genes function through this pathway requires further research.
OsERF83 improves drought tolerance in rice but also causes growth inhibition and reduced yield [28]. AmDREB1F increases the drought tolerance of Arabidopsis, but constitutive expression also leads to a phenotype of delayed growth [29].OsTZF5 improves the survival rate of rice under drought stress and leads to growth inhibition [3].The constitutive expression of GmNAC085 in Arabidopsis improves drought tolerance and also results in delayed growth of aboveground and root [30].These transcription factors all enhance plant drought resistance while causing plant growth inhibition.JcZFP8 enhanced tobacco drought resistance and also caused delayed germination and early growth inhibition.We also used qRT-PCR to determine the expression of some NAC, ZFP, and ERF transcription factors in NAC, ZFP, and the co-overexpression line (Figure 2e).While ZFP overexpression improved tolerance to osmotic stress (Figure 1k,l), ZFP alone did not significantly upregulate the tested stress-related transcription factors (Figure 2e).Zinc finger proteins in Arabidopsis are involved in enhancing stress tolerance, but often have a negative effect on growth [10].In ZFPL-overexpressing plants, the expression of photosynthesis-related genes was downregulated, resulting in plant growth inhibition [31].In the current study, the overexpression of JcZFP8 transiently maintained photosynthetic rate on day 2 after drought stress treatment (Figure 1g).In previous studies, JcZFP8 affected the growth of Nicotiana tabacum L. via GA, resulting in plant dwarfing [21].It is possible that other types of transcription factors are regulated by JcZFP8, resulting in ZFP improving plant drought resistance and inhibiting growth.
ZFP exhibited growth inhibition at three weeks of age, but there was no significant difference between the double gene co-expression line NZ and WT (Figures 1h and S1).The NAC protein is located at the branching point of the ABA-dependent and independent pathway, which can avoid cross talk between stress resistance genes and growth genes [32].The interaction between the GA inhibitor DELLA and NAC protein in cotton mediates GA signal transduction [33].ScNAC23 can accelerate GA-mediated flowering and senescence in Arabidopsis [34].JcZFP8 inhibits plant growth and development via GA, whereas JcNAC1 can regulate plant flowering and premature senescence by enhancing GA signaling.JcNAC1 and JcZFP8 may work antagonistically via GA to regulate the growth of NZ, avoiding early flowering and senescence caused by JcNAC1, accelerating growth inhibition caused by JcZFP8 and ensuring the agronomic advantage of NZ.
Plants 2023, 12, 3029 9 of 14 NAC, ZFP, and NZ accumulated less reactive oxygen species and more proline under osmotic stress, endowing plants with stronger osmotic stress tolerance (Figures 1d-g and  2a-d).Under drought stress, CarNAC4 in Arabidopsis resulted in lower MDA and low water loss [35].SlNAC10 improved drought tolerance by increasing proline synthesis [36].The gene expression in jasmonic acid (JA) synthesis and the signaling of VaNAC17-expressing Arabidopsis plants is upregulated under drought stress, resulting in the reduced accumulation of reactive oxygen species in plants [8].The overexpression of ZF2 in Arabidopsis enhanced drought tolerance by increasing proline [37].The overexpression of OsDRZ1 in rice improves plant drought resistance by reducing ROS, and increasing proline [10].These studies suggest that NAC and C2H2-ZFP transcription factors can regulate drought resistance by reducing ROS accumulation, increasing proline content, and regulating the expression of downstream jasmonic acid response genes.We found that NAC, ZFP, and NZ improved resistance to drought stress in these ways.The content of O 2− and H 2 O 2 in NZ was lower, but NZ had significantly increased proline content, which ensured stronger tolerance to drought stress in NZ.
Our results demonstrated that the drought resistance of the double-gene transformed lines was stronger than that of the single-gene transformed lines, and the drought resistance was superimposed in the double-gene transformed lines.PpNAC2 and PpNAC3 were related to the JA response in pine [38].AtZP1 regulates genes encoding bHLH transcription factors [39].NbbHLH1 and NbbHLH2 were upregulated in transgenic plants; in addition, superimposed expression was found in the double gene co-expressed plants.These results indicate that these two transcription factors are involved in the increasing drought resistance of transgenic plants.
Under drought stress, the mutant tobacco bhlh1, bhlh2, and bhlh1bhlh2 accumulated more H 2 O 2 and MDA (Figure 3d,e).MYC2 was involved in plant drought tolerance associated with JA [40].PxbHLH02 increased the drought tolerance of poplar with lower H 2 O 2 [41].MdCIB1-transgenic Arabidopsis exhibited drought tolerance with lower MDA and H 2 O 2 accumulation [42].NbbHLH1 and NbbHLH2 were positive regulators in the JA pathway [11].The JAZ-MYC module was a central component of JA response [43].Our studies provide evidence that NbbHLH1 and NbbHLH2 may not regulate the expression of NbMYB21 and NbMYB86, Further research is needed on how NbbHLH1 and NbbHLH2 regulate tobacco drought resistance through the JA pathway.
After identification and sequencing, the correct recombinant vectors were transformed into Agrobacterium GV3101, which was used for genetic transformation as previously described [44].Positive transgenic plants were selected as follows: Single gene overexpression JcNAC1 lines (named NAC) by hygromycin (50 mg/L), Single gene overexpression JcZFP8 lines (named ZFP) by kanamycin (50 mg/L) and the double-gene overexpression JcNAC1-JcZFP8 lines (named NZ) by basta (20 mg/L).The expression of JcNAC1 and JcZFP8 in T 1 were verified by PCR and qRT-PCR (Figure S1b).Subsequent experiments were performed using T 2 tobacco.
All primers were synthesized by Sangon (China), the primer sequences are shown in (Table S7).The sequences of NbbHLH1 (NbS00019773g0102) and NbbHLH2 (NbS00001919g0002) were extracted from previous studies [12].sgRNA for NbbHLH1 and NbbHLH2 were designed on the website https://crispr.dbcls.jp/(last access date 13 August 2023), and different knockout vectors of NbbHLH1 and NbbHLH2 were constructed on PHSE401 [45].The vector was transformed into Agrobacterium GV3101 and then used for genetic transformation as described in 2.1 (Figure S5).Knocked-out lines of NbbHLH1 (named bhlh1) and knocked-out lines of NbbHLH2 (named bhlh2) were selected by hygromycin (50 mg/L).The knockout results in T 1 were validated by PCR and sequencing (Figure S4a,b).We selected T 2 bhlh1 and bhlh2, which can only be knocked out at one site, for subsequent experiments.
Based on the knockout results of bhlh1 and bhlh2, we selected sgRNA for NbbHLH1 and NbbHLH2 to construct the knockout vector (Figure S5) and obtained double knockedout lines (named bhlh1bhlh2) using the method described above.Double knocked-out lines of bhlh1bhlh2 were selected by hygromycin (50 mg/L).We selected T 2 bhlh1bhlh2 for subsequent experiments.

Plant Materials and Drought Stress Treatments
Nicotiana benthamiana L. was used in this study.Seeds were sterilized with 75% alcohol for 3 min and planted on 1/2 Murashige Skoog (MS) medium (1/2 MS medium containing 3% sucrose and 0.7% agar).Seven days later, tobacco was planted in the pot (Diameter × Bottom diameter × Height: 90 mm × 60 mm × 75 mm) full of soil (nutrient soil: vermiculite = 1:1) at 25 • C with a light density of ~120 µmol m −2 s −1 under a photoperiod of 16/8 h in greenhouse.
WT and transgenic tobacco "JcNAC1(NAC), JcZFP8 (ZFP) and JcNAC1-JcZFP8(NZ)" at five weeks of age were stressed with 10% PEG6000 for 4 days.WT and knockout tobacco "bhlh1, bhlh2 and bhlh1bhlh2" at five weeks of age were stressed with withholding watering for 7 days.The second fully unfolded leaf from the top to the bottom was selected, frozen in liquid nitrogen for 30 min, and stored in a −80 • C low-temperature refrigerator for use.

Germination Rates and Other Growth Trait
Sterilized seeds of WT, overexpression lines (NAC, ZFP and NZ), and knockout lines (bhlh1, bhlh2, and bhlh1bhlh2) were placed on 1/2 MS medium with mannitol (0 mM, 150 mM, 200 mM).The seed germination rates were counted and assayed with three biological replicates.Germination index = ∑ , n i is the number of germinated seeds on the ith day (t i ) in percentage by days −1 [46].

Flowering Period, Life Cycle, Weight, and Yield
Flowering time was defined as full expansion of the first flower.Life cycle was defined as the first upper leaf turning completely yellow.After the plant was fully harvested, the above-ground parts were oven-dried at 70 • C for 3 days and the seeds were oven-dried at 37 • C for 7 days and weighted.Twenty-four biological replicates of each sample were collected for analysis [47].

Expression Analysis of Important Genes in Drought Treatments
Plant leaves were stored in −80 • C low-temperature refrigerator for use in Section 2.3.Total RNA was extracted using Kit from Foregene (Chengdu, China).The cDNA synthesized and qPCR performed using Kit from Vazyme (Nanjing, China), all primers were synthesized by Sangon (Shanghai, China), the primer sequences are shown in (Table S1), and the reaction system and procedure were performed according to Shi et al. [21].Three biological replicates were prepared for each plant group.

Measurements of Physiological Parameters
Fresh leaves of WT, NAC, ZFP, and NZ lines were collected after 10% PEG6000 treatments for 4 days for measurement of O 2− and H 2 O 2 , malondialdehyde (MDA), and proline level using the related test kits (Solarbio, Beijing, China); we followed the instructions of the reagent kit for specific operations.[49].
Fresh leaves of WT, bhlh1, bhlh2, and bhlh1bhlh2 knockout lines at five weeks of age were stressed with withholding watering for 7 days for measurement of physiological parameters related to drought, including relative water content (RWC), MDA, and H 2 O 2 [50].
The second fully unfolded leaf from the top to the bottom was selected with three biological replicates.RWC = (FW − DW)/(TW − DW) × 100% (FW: fresh weight; DW: dry weight; TW: turgid fresh weight).

Conclusions
In this study, the co-expression of two different transcription factors JcNAC1 and JcZFP8 in tobacco demonstrated better growth and drought resistance traits.Under 10% PEG6000 treatment, transgenic plants, especially the double overexpression line NZ, showed better drought tolerance, accumulating more proline and less H 2 O 2 and less MDA.In transgenic plants.NbbHLH1 and NbbHLH2 manifested a superimposed effect, which played significant roles in the increased drought resistance of transgenic plants.Under drought treatment, the drought resistance of the knock-out lines bhlh1, bhlh2, and bhlh1bhlh2 was weakened, and more H 2 O 2 and MDA were accumulated.We provided evidence that NbbHLH1 and NbbHLH2 might not regulate the drought response by affecting the expression of NbMYB21 and NbMYB86 in the JA pathway.Our findings can provide guidance on theory and selection of gene combinations for application of multi-gene co-expression in agriculture in the future.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12173029/s1,Figure: S1 Growth traits of three weeks of WT and overexpression lines;  S1: Germination rate of overexpressed tobacco under Mannitol germination stress.;Table S2: Growth traits of WT and overexpression lines 4; Table S3: Leaf gas exchange traits of WT and overexpression lines under PEG-drought; Table S4: Effect of singlegene overexpression (NAC and ZFP) versus double gene co-expression (NZ) on tolerance to 10% PEG6000 osmotic stress treatment.;Table S5: Germination rate of knock-out tobacco under Mannitol germination stress; Table S6: Effect of knock-out tobacco on tolerance drought stress; Table S7: Primers used in this study.
Author Contributions: Y.X., F.C. and X.N. conceived and designed the experiments; X.N. performed most experiments; The first draft of the manuscript was written by X.N. and all authors commented on previous versions of the manuscript.X.N. and Z.L. performed expression analysis.L.W. Provide plant materials.Y.R., R.M., X.W., C.C. and T.W. performed physiological and biochemical indices measures.All authors have read and agreed to the published version of the manuscript.

Figure 1 .
Figure 1.Growth and leaf gas exchange traits of WT and overexpression lines.(a) Germination rate of seeds treated with different mannitol concentrations; (b) germination rate in 1/2 MS medium; (c) germination index in 1/2 MS medium; (d) plant appearance after four days of 10%PEG6000

Figure 2 .
Figure 2. The effect of single-gene overexpression (NAC and ZFP) versus double gene co-expression (NZ) on tolerance to 10% PEG6000 osmotic stress treatment.(a) O 2− content; (b) H2O2 content; (c) Proline content; (d) MDA content; (e) transcription factor regulation involved in tobacco drought response.The superscript letters in the table represent the differences between different genotypes, and different letters indicate significant differences (p < 0.05), data are means ± SEM.

Figure 2 .
Figure 2. The effect of single-gene overexpression (NAC and ZFP) versus double gene co-expression (NZ) on tolerance to 10% PEG6000 osmotic stress treatment.(a) O 2− content; (b) H 2 O 2 content; (c) Proline content; (d) MDA content; (e) transcription factor regulation involved in tobacco drought response.The superscript letters in the table represent the differences between different genotypes, and different letters indicate significant differences (p < 0.05), data are means ± SEM.

14 Figure 3 .
Figure 3.The effect of bhlh1, bhlh2, and bhlh1bhlh2 knock-out on properties related to drought response.Properties in panels a and b were in plants subjected to mannitol osmotic stress; properties in panels c to g were measured on leaves of pot-grown plants that were subjected to drought by withholding water for 7 days.(a) germination and growth of bhlh1, bhlh2, and bhlh1bhlh2 in control and mannitol medium; (b) germination rate of bhlh1, bhlh2, and bhlh1bhlh2 under mannitol treatment; (c) relative water content of bhlh1, bhlh2, and bhlh1bhlh2; (d) H2O2 content; (e) MDA content; (f) expression of genes in the JA signaling pathway; and (g) gene expression of MYB transcription factors.The superscript letters in the table represent the differences between different genotypes, and different letters indicate significant differences (p < 0.05), data are means ± SEM.

Figure 3 .
Figure 3.The effect of bhlh1, bhlh2, and bhlh1bhlh2 knock-out on properties related to drought response.Properties in panels a and b were in plants subjected to mannitol osmotic stress; properties in panels

2. 3 . 3 .
NbbHLH1 and NbbHLH2 May Not Affect Drought Resistance by Regulating the Expression of NbMYB21 and NbMYB86

4. 2 .
Construction and Transformation of NbbHLH1, NbbHLH2, and NbbHLH1-NbbHLH2 Figure S2: ROS of single-gene overexpression (NAC and ZFP) versus double gene co-expression (NZ) on tolerance to 10% PEG6000 osmotic stress treatment; Figure S3: Construction of overexpressed plants and Map of genes overexpression vector; Figure S4: Sequence analysis of NbbHLH1 and NbbHLH2 gene knockout tobacco; Figure S5: Schematic diagram of double target vector construction; Table /JcZFP8 Not Only Improves PEG-Drought Resistance, but Also Overcomes the Adverse Agronomic Traits Brought by Single Gene Transformation 2.1.1.Co-Expression of JcNAC1 and JcZFP8 Can Increase the Germination Rate of Plants under Mannitol-Drought to g were measured on leaves of pot-grown plants that were subjected to drought by withholding water for 7 days.(a) germination and growth of bhlh1, bhlh2, and bhlh1bhlh2 in control and mannitol medium; (b) germination rate of bhlh1, bhlh2, and bhlh1bhlh2 under mannitol treatment; (c) relative water content of bhlh1, bhlh2, and bhlh1bhlh2; (d) H 2 O 2 content; (e) MDA content; (f) expression of genes in the JA signaling pathway; and (g) gene expression of MYB transcription factors.The superscript letters in the table represent the differences between different genotypes, and different letters indicate significant differences (p < 0.05), data are means ± SEM. c